A technique to study the link between physiological aging and disease, from the Institute of Photonic Sciences (ICFO), uses single-laser-based optical tweezers to measure the viscoelasticity of biological materials as they mature. In biology, changes in viscoelasticity can lead to cancer, neurodegenerative disorders, and other diseases. Knowledge of the viscoelasticity and other rheological properties of intracellular organelles, cells, and whole tissues is central to understanding their physiological functions. Knowledge of how changes to viscoelasticity affect cellular health could help speed diagnosis and drug development. Optical tweezers are well-suited to obtaining the material properties of biological materials because they operate in the piconewton (pN) range — the typical range for molecular interactions — and can measure the position of micrometer-sized objects with sub-nanometer accuracy by using noninvasive IR laser light. However, optical tweezers require a complicated, costly experimental setup, including a perfectly aligned dual laser system that only a few research sites worldwide can accommodate. The ICFO team, in collaboration with Impetux Optics, the Center of Genomic Regulation, the Institute for Research in Biomedicine, and Universitat Pompeu Fabra, developed a method to characterize the microrheological properties of samples using time-shared optical tweezers, which are less expensive and less complex than conventional tweezers. The microrheology method based on time-shared optical tweezers, called TimSOM, simplifies the optical setup and enhances the versatility of the tweezers-based technique. “At the same time, TimSOM is accompanied by a step-by-step protocol on how to use it, which will facilitate the adoption of optical tweezers-based microrheology in the fields of molecular, cellular, and developmental biology,” professor Michael Krieg said. (From left) Neus Sanfeliu, Santiago Ortiz, Martín Fernández, Frederic Català, and Michael Krieg worked on the development of time-shared optical tweezers to measure the viscoelasticity of biological materials as they mature. Courtesy of ICFO. With TimSOM, a single laser beam is split into two near-instantaneous, time-shared optical traps — one for driving active oscillations and the other for static displacement detection. In addition to performing simultaneous force and displacement measurements, TimSOM quantifies the mechanical properties of the sample, ranging from millipascals to kilopascals across five decades of frequency. The use of a single laser for force and position measurements eliminates the challenge of aligning two optical paths and two detectors. It also reduces costs and measurement time. “Because we used the same laser, our measurements were easy to conduct at different locations within the same living cells, which otherwise are notoriously difficult to perform,” researcher Frederic Catala-Castro said. “In other words, the single laser optical trap can be displaced at any position in the field of view, which enhances the spatiotemporal versatility of this method.” However, there was one drawback to using a single, time-shared laser for optical tweezing. “Time-sharing means that the same laser half of the time measures the force, and the other half measures the particle displacement due to this force,” researcher Paolo-Antonio Frigeri said. Consequently, the stress and strain measurements occur quasi-simultaneously, but not at the exact same time. To address this issue, the team developed a theoretical framework to obtain the viscoelastic parameters and retrieve the missing data from the raw measurements. To create a practical, robust nanorheometer, the researchers analyzed typical deviations from the ideal behavior using numerical and analytical models, and offered solutions to account for these discrepancies. Through experiments on the rheological properties of biomolecular condensates, cells, and animals, the researchers demonstrated the versatility of TimSOM and explored the relationship between material properties, morphogenesis, aging, and disease. The researchers observed the viscoelasticity inside a protein condensate, which is known to undergo an age-dependent transition from a liquid to a more solid state. Such phase transitions are thought to be related to neurodegenerative diseases. The team demonstrated TimSOM on live cells isolated from zebrafish embryos and quantified the complex viscoelastic properties of intracellular compartments of the zebrafish progenitor cells. The researchers also explored the relation between viscoelasticity and aging in the intestinal tissues of C. elegans. TimSOM showed how mutations in nuclear envelope proteins, which cause premature aging disorders in humans, soften the cytosol of C. elegans intestinal cells during the aging of the organism. The researchers demonstrated that time-shared optical tweezer microrheology could provide rapid phenotyping of material properties inside cells and protein blends, which could be useful for biomedical and drug-screening applications and for fields involving material characterization. Because TimSOM requires only a small amount of a sample, it could be used to quantify the rheological properties of precious and rare materials. It is suitable for industrial applications in, for example, the food processing, cosmetics, and pharmaceutical industries, which need to characterize emulsions, blends, protein droplets, and other biologically derived liquids. However, Krieg is even more excited about TimSOM’s potential to help uncover the answers to fundamental scientific questions. “Which is the energy that a cell requires in order to move? How does the nucleus protect DNA and activate transcription? How does the deformation of a mechanosensitive protein condensate translate into the activation of a neuron? “TimSOM will help scientists in the field take a picture of biological mechanics, a stiffness map of a biomaterial, and luckily that might allow us to finally answer these and many other long-lived questions in rheology,” he said. The research was published in Nature Nanomaterials (www.doi.org/10.1038/s41565-024-01830-y).